Enhancer switching in cell lineage priming is linked to eRNA, Brg1's AT-hook, and SWI/SNF recruitment.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.

MED1 is a lipogenesis coactivator required for postnatal adipose expansion.

MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro, but its role in adipose development and expansion in vivo has not been reported. Here, we show that MED1 is not generally required for transcription during adipogenesis in culture and that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion and the induction of fatty acid and triglyceride synthesis genes after pups switch diet from high-fat maternal milk to carbohydrate-based chow. During adipogenesis, MED1 is dispensable for induction of lineage-determining transcription factors (TFs) PPARγ and C/EBPα but is required for lipid accumulation in the late phase of differentiation. Mechanistically, MED1 controls the induction of lipogenesis genes by facilitating lipogenic TF ChREBP- and SREBP1a-dependent recruitment of Mediator to active enhancers. Together, our findings identify a cell- and gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion.

Critical roles of transcriptional coactivator MED1 in the formation and function of mouse adipose tissues.

The MED1 subunit has been shown to mediate ligand-dependent binding of the Mediator coactivator complex to multiple nuclear receptors, including the adipogenic PPARγ, and to play an essential role in ectopic PPARγ-induced adipogenesis of mouse embryonic fibroblasts. However, the precise roles of MED1, and its various domains, at various stages of adipogenesis and in adipose tissue have been unclear. Here, after establishing requirements for MED1, including specific domains, for differentiation of 3T3L1 cells and both primary white and brown preadipocytes, we used multiple genetic approaches to assess requirements for MED1 in adipocyte formation, maintenance, and function in mice. We show that MED1 is indeed essential for the differentiation and/or function of both brown and white adipocytes, as its absence in these cells leads to, respectively, defective brown fat function and lipodystrophy. This work establishes MED1 as an essential transcriptional coactivator that ensures homeostatic functions of adipocytes.

Med1 controls thymic T-cell migration into lymph node through enhancer-based Foxo1-Klf2 transcription program.

The migration is the key step for thymic T cells to enter circulation and then lymph nodes (LNs), essential for future immune surveillance. Although promoter-based transcriptional regulation through Foxo1, Klf2, Ccr7, and Sell regulates T-cell migration, it remains largely unexplored whether and how enhancers are involved in this process. Here we found that the conditional deletion of Med1, a component of the mediator complex and a mediator between enhancers and RNA polymerase II, caused a reduction of both CD4+ and CD8+ T cells in LNs, as well as a decrease of CD8+ T cells in the spleen. Importantly, Med1 deletion hindered the migration of thymic αßT cells into the circulation and then into LNs, accompanied by the downregulation of KLF2, CCR7, and CD62L. Mechanistically, Med1 promotes Klf2 transcription by facilitating Foxo1 binding to the Klf2 enhancer. Furthermore, forced expression of Klf2 rescued Ccr7 and Sell expression, as well as αßT-cell migration into LNs. Collectively, our study unveils a crucial role for Med1 in regulating the enhancer-based Foxo1-Klf2 transcriptional program and the migration of αßT cells into LNs, providing valuable insights into the molecular mechanisms underlying T-cell migration.

MED1 Regulates BMP/TGF-ß in Endothelium: Implication for Pulmonary Hypertension.

BACKGROUND: Dysregulated BMP (bone morphogenetic protein) or TGF-ß (transforming growth factor beta) signaling pathways are imperative in idiopathic and familial pulmonary arterial hypertension (PAH) as well as experimental pulmonary hypertension (PH) in rodent models. MED1 (mediator complex subunit 1) is a key transcriptional co-activator and KLF4 (Krüppel-like factor 4) is a master transcription factor in endothelium. However, MED1 and KLF4 epigenetic and transcriptional regulations of the BMP/TGF-ß axes in pulmonary endothelium and their dysregulations leading to PAH remain elusive. We investigate the MED1/KLF4 co-regulation of the BMP/TGF-ß axes in endothelium by studying the epigenetic regulation of BMPR2 (BMP receptor type II), ETS-related gene (ERG), and TGFBR2 (TGF-ß receptor 2) and their involvement in the PH. METHODS: High-throughput screening involving data from RNA-seq, MED1 ChIP-seq, H3K27ac ChIP-seq, ATAC-seq, and high-throughput chromosome conformation capture together with in silico computations were used to explore the epigenetic and transcriptional regulation of BMPR2, ERG, and TGFBR2 by MED1 and KLF4. In vitro experiments with cultured pulmonary arterial endothelial cells (ECs) and bulk assays were used to validate results from these in silico analyses. Lung tissue from patients with idiopathic PAH, animals with experimental PH, and mice with endothelial ablation of MED1 (EC-MED1-/-) were used to study the PH-protective effect of MED1. RESULTS: Levels of MED1 were decreased in lung tissue or pulmonary arterial endothelial cells from idiopathic PAH patients and rodent PH models. Mechanistically, MED1 acted synergistically with KLF4 to transactivate BMPR2, ERG, and TGFBR2 via chromatin remodeling and enhancer-promoter interactions. EC-MED1-/- mice showed PH susceptibility. In contrast, MED1 overexpression mitigated the PH phenotype in rodents. CONCLUSIONS: A homeostatic regulation of BMPR2, ERG, and TGFBR2 in ECs by MED1 synergistic with KLF4 is essential for the normal function of the pulmonary endothelium. Dysregulation of MED1 and the resulting impairment of the BMP/TGF-ß signaling is implicated in the disease progression of PAH in humans and PH in rodent models.

Mediator subunit MED1 is required for E2A-PBX1-mediated oncogenic transcription and leukemic cell growth.

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1-dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1-driven leukemia. The MED1 dependency for E2A-PBX1-mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

Mediator subunit MED1 deficiency prevents carbon tetrachloride-induced hepatic fibrosis in mice.

Mediator subunit mediator 1 (MED1) mediates ligand-dependent binding of the mediator coactivator complex to various nuclear receptors and plays a critical role in embryonic development, lipid and glucose metabolism, liver regeneration, and tumorigenesis. However, the precise role of MED1 in the development of liver fibrosis has been unclear. Here, we showed that MED1 expression was increased in livers from nonalcoholic steatohepatitis (NASH) patients and mice and positively correlated with transforming growth factor ß (TGF-ß) signaling and profibrotic factors. Upon treatment with carbon tetrachloride (CCl4), hepatic fibrosis was much less in liver-specific MED1 deletion (MED1ΔLiv) mice than in MED1fl/fl littermates. TGF-ß/Smad2/3 signaling pathway was inhibited, and gene expression of fibrotic markers, including α-smooth muscle actin (α-SMA), collagen type 1 α 1 (Col1a1), matrix metalloproteinase-2 (Mmp2), and metallopeptidase inhibitor 1 (Timp1) were decreased in livers of MED1ΔLiv mice with CCl4 injection. Transcriptomic analysis revealed that the differentially expressed genes in livers of CCl4-administered MED1ΔLiv mice were enriched in the pathway of oxidoreductase activity, followed by robustly reduced oxidoreductase activity-related genes, such as Gm4756, Txnrd3, and Etfbkmt. More importantly, we found that the reduction of reactive oxygen species (ROS) in MED1 knockdown hepatocytes blocked the activation of TGF-ß/Smad2/3 pathway and the expression of fibrotic genes in LX2 cells. These results indicate that MED1 is a positive regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for the regression of liver fibrosis.NEW & NOTEWORTHY In this study, we present the first evidence that liver mediator 1 (MED1) deficiency attenuated carbon tetrachloride-induced hepatic fibrosis in mouse. The underlying mechanism is that MED1 deficiency reduces reactive oxygen species (ROS) production in hepatocytes, thus restricts the activation of TGF-ß/Smad2/3 signaling pathway and fibrogenic genes expression in hepatic stellate cells (HSCs). These data suggest that MED1 is an essential regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for liver fibrosis.

PRDM16 binds MED1 and controls chromatin architecture to determine a brown fat transcriptional program.

PR (PRD1-BF1-RIZ1 homologous) domain-containing 16 (PRDM16) drives a brown fat differentiation program, but the mechanisms by which PRDM16 activates brown fat-selective genes have been unclear. Through chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analyses in brown adipose tissue (BAT), we reveal that PRDM16 binding is highly enriched at a broad set of brown fat-selective genes. Importantly, we found that PRDM16 physically binds to MED1, a component of the Mediator complex, and recruits it to superenhancers at brown fat-selective genes. PRDM16 deficiency in BAT reduces MED1 binding at PRDM16 target sites and causes a fundamental change in chromatin architecture at key brown fat-selective genes. Together, these data indicate that PRDM16 controls chromatin architecture and superenhancer activity in BAT.

PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex.

MED1/BDNF/TrkB pathway is involved in thalamic hemorrhage-induced pain and depression by regulating microglia.

Central post-stroke pain (CPSP) and associated depression remain poorly understood and pharmacological treatments are unsatisfactory. Recently, microglia activation was suggested to be involved in CPSP pathophysiology. The goal of this study was to investigate the effectiveness of a co-ultramicronized combination of N-palmitoylethanolamide and luteolin (PEALut) in a mouse model of thalamic hemorrhage (TH)-induced CPSP. TH was established through the collagenase-IV injection in thalamic ventral-posterolateral-nucleus. PEALut effects in CPSP-associated behaviors were evaluated during a 28-days observation period. We found that repeated administrations of co-ultra PEALut significantly reduced mechanical hypersensitivity after TH, as compared to vehicle, by reducing the early microglial activation in the perilesional site. Moreover, PEALut prevented the development of depressive-like behavior (21 days post-TH). These effects were associated with the restoration of synaptic plasticity in LEC-DG pathway and monoamines levels found impaired in TH mice. Hippocampal MED1 and TrkB expressions were significantly increased in TH compared to sham mice 21 days post-TH, whereas BDNF levels were decreased. PEALut restored MED1/TrkB/BDNF expression in mice. Remarkably, we found significant overexpression of MED1 in the human autoptic brain specimens after stroke, indicating a translational potential of our findings. These results pave the way for better-investigating depression in TH- induced CPSP, together with the involvement of MED1/TrkB/BDNF pathway, proposing PEALut as an adjuvant treatment.

MED1 Ablation Promotes Oral Mucosal Wound Healing via JNK Signaling Pathway.

Mediator complex subunit 1 (MED1) is a coactivator of multiple transcription factors and plays a key role in regulating epidermal homeostasis as well as skin wound healing. It is unknown, however, whether it plays a role in healing oral mucosal wounds. In this study, we investigate MED1's functional effects on oral mucosal wound healing and its underlying mechanism. The epithelial-specific MED1 null (Med1epi-/-) mice were established using the Cre-loxP system with C57/BL6 background. A 3 mm diameter wound was made in the cheek mucosa of the 8-week-old mice. In vivo experiments were conducted using HE staining and immunostaining with Ki67 and uPAR antibodies. The in vitro study used lentiviral transduction, scratch assays, qRT-PCR, and Western blotting to reveal the underlying mechanisms. The results showed that ablation of MED1 accelerated oral mucosal wound healing in 8-week-old mice. As a result of ablation of MED1, Activin A/Follistatin expression was altered, resulting in an activation of the JNK/c-Jun pathway. Similarly, knockdown of MED1 enhanced the proliferation and migration of keratinocytes in vitro, promoting re-epithelialization, which accelerates the healing of oral mucosal wounds. Our study reveals a novel role for MED1 in oral keratinocytes, providing a new molecular therapeutic target for accelerated wound healing.

Med1 controls CD8 T cell maintenance through IL-7R-mediated cell survival signalling.

Under steady-state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8+ T cells compared with WT counterparts. Finally, Med1-deficient CD8+ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.

Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo.

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit-TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

Extracellular vesicle-encapsulated microRNA-425-derived from drug-resistant cells promotes non-small-cell lung cancer progression through DAPK1-medicated PI3K/AKT pathway.

Investigations in the area of tumor-derived extracellular vesicles (EVs) open a new horizon in developing cancer biology and its potential as cancer biomarkers. Following this prospect, we aimed to identify that the role of successfully isolated EVs from drug-resistance cells in the progression of non-small-cell lung cancer (NSCLC). P-EVs and R-EVs secreted by A549 cells and drug-resistant A549-R cells respectively were extracted and characterized. The targeting relationship between miR-425 and MED1 was verified. Cell proliferation, invasion, migration and apoptosis after treatment of P-EVs, R-EVs, miR-425 inhibitor, miR-425 mimic, pcDNA-MED1, or phosphatidylinositol-3-kinase (PI3K)/AKT inhibitor LY294002 were detected. Furthermore, xenograft tumor in nude mice was established for further confirming our in vitro findings. P-EVs and R-EVs were successfully extracted and could be internalized by A549 cells. A549-R cells and R-EVs showed higher miR-425 expression compared with A549 cells and P-EVs, respectively. miR-425 delivered by R-EVs could promote the proliferation, migration, and invasion, while inhibit apoptosis of NSCLC cells. MED1 was the target gene of miR-425. EVs-encapsulated miR-425-derived from A549-R cells could promote the progression of NSCLC in vivo through regulating DAPK1-medicated PI3K/AKT pathway. Moreover, miR-425 delivered by R-EVs promoted tumorigenesis in vivo. Taken together, the result suggested that EVs-delivered miR-425-derived from A549-R cells promoted the progression of NSCLC through regulating DAPK1-medicated PI3K/AKT signaling pathway.

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor.

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3 )-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRß1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRß1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRß1 population were significantly greater in MED1-/- cells, compared with MED1+/+ cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRß1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.

The transcription factor FgMed1 is involved in early conidiogenesis and DON biosynthesis in the plant pathogenic fungus Fusarium graminearum.

Fusarium graminearum is a prominent fungal pathogen that causes economically important losses by infesting a wide variety of cereal crops. F. graminearum produces both asexual and sexual spores which disseminate and inoculate hosts. Therefore, to better understand the disease cycle and to develop strategies to improve disease management, it is important to further clarify molecular mechanisms of F. graminearum conidiogenesis. In this study, we functionally characterized the FgMed1, a gene encoding an ortholog of a conserved MedA transcription factor known to be a key conidiogenesis regulator in Aspergillus nidulans. The gene deletion mutants ΔFgMed1 produced significantly less conidia, and these were generated from abnormal conidiophores devoid of phialides. Additionally, we observed defective sexual development along with reduced virulence and deoxynivalenol (DON) production in ΔFgMed1. The GFP-tagged FgMed1 protein localized to the nuclei of conidiophores and phialides during early conidiogenesis. Significantly, RNA-Seq analyses showed that a number of the conidiation- and toxin-related genes are differentially expressed in the ΔFgMed1 mutant in early conidiogenesis. These data strongly suggest that FgMed1 involved in regulation of genes associated with early conidiogenesis, DON production, and virulence in F. graminearum.

MicroRNA-146a protects against myocardial ischaemia reperfusion injury by targeting Med1.

BACKGROUND: Myocardial ischaemia reperfusion injury (MIRI) is a difficult problem in clinical practice, and it may involve various microRNAs. This study investigated the role that endogenous microRNA-146a plays in myocardial ischaemia reperfusion and explored the possible target genes. METHODS: MIRI models were established in microRNA-146a deficient (KO) and wild type (WT) mice. MicroRNA-146a expression was evaluated in the myocardium of WT mice after reperfusion. The heart function, area of myocardium infarction and in situ apoptosis were compared between the KO and WT mice. Microarray was used to explore possible target genes of microRNA-146a, while qRT-PCR and dual luciferase reporter assays were used for verification. Western blotting was performed to detect the expression levels of the target gene and related signalling molecules. A rescue study was used for further testing. RESULTS: MicroRNA-146a was upregulated 1 h after reperfusion. MicroRNA-146a deficiency decreased heart function and increased myocardial infarction and apoptosis. Microarray detected 19 apoptosis genes upregulated in the KO mice compared with the WT mice. qRT-PCR and dual luciferase verified that Med1 was one target gene of microRNA-146a. TRAP220, encoded by Med1 in the KO mice, was upregulated, accompanied by an amplified ratio of Bax/Bcl2 and increased cleaved caspase-3. Inhibition of microRNA-146a in H9C2 cells caused increased TRAP220 expression and more apoptosis under the stimulus of hypoxia and re-oxygenation, while knockdown of the increased TRAP220 expression led to decreased cell apoptosis. CONCLUSIONS: MicroRNA-146a exerts a protective effect against MIRI, which might be partially mediated by the target gene Med1 and related to the apoptosis signalling pathway.

Mouse Cardiac Pde1C Is a Direct Transcriptional Target of Pparα.

Phosphodiesterase 1C (PDE1C) is expressed in mammalian heart and regulates cardiac functions by controlling levels of second messenger cyclic AMP and cyclic GMP (cAMP and cGMP, respectively). However, molecular mechanisms of cardiac Pde1c regulation are currently unknown. In this study, we demonstrate that treatment of wild type mice and H9c2 myoblasts with Wy-14,643, a potent ligand of nuclear receptor peroxisome-proliferator activated receptor alpha (PPARα), leads to elevated cardiac Pde1C mRNA and cardiac PDE1C protein, which correlate with reduced levels of cAMP. Furthermore, using mice lacking either Pparα or cardiomyocyte-specific Med1, the major subunit of Mediator complex, we show that Wy-14,643-mediated Pde1C induction fails to occur in the absence of Pparα and Med1 in the heart. Finally, using chromatin immunoprecipitation assays we demonstrate that PPARα binds to the upstream Pde1C promoter sequence on two sites, one of which is a palindrome sequence (agcTAGGttatcttaacctagc) that shows a robust binding. Based on these observations, we conclude that cardiac Pde1C is a direct transcriptional target of PPARα and that Med1 may be required for the PPARα mediated transcriptional activation of cardiac Pde1C.

Checkpoint-dependent phosphorylation of Med1/TRAP220 in response to DNA damage.

Mediator complex subunit 1 (Med1)/Thyroid hormone receptor-associated protein 220 (TRAP220), an essential component of thyroid hormone receptor-associated proteins (TRAP)/mediator, plays important roles in hormone responses and tumorigenesis. However, the role of Med1 in the DNA damage response has not been studied. In this study, we found that DNA damage, resulted from γ-irradiation, ultraviolet (UV)-irradiation, or hydroxyurea, induced phosphorylation of Med1 in vivo. Phosphorylation of Med1 was abrogated by either caffeine or wortmannin treatment, suggesting that Med1 is phosphorylated through the DNA damage checkpoint pathway. A checkpoint kinase 1 (Chk1)/checkpoint kinase 2 (Chk2) consensus phosphorylation motif was identified at Serine 671 of Med1 and Ser671 motif was primarily phosphorylated by Chk2 in vitro. Moreover, the in vivo phosphorylation of Med1 was abrogated by a Chk2 inhibitor, and physical interaction between Chk2 and Med1 was observed, confirming that Chk2 is responsible for Med1 phosphorylation upon DNA damage. These results suggest that Med1 is a novel target for the DNA damage checkpoint pathway and may participate in the DNA damage response. Consistent with this notion, knockdown of Med1 expression caused a significant increase in cellular sensitivity to UV irradiation. Moreover, microarray analysis revealed that the UV-induced activation of the transcription of important regulators of cell cycle control and DNA repair, including p21, Gadd45, Rad50, DnaJ, and RecQL, was impaired upon Med1 knockdown. Taken together, our data suggest that Med1 is a novel target for Chk2-mediated phosphorylation and may play a role in cellular DNA damage responses by mediating proper induction of gene transcription upon DNA damage.

Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation.

The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer-promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 (R(2) = 0.6213, P < 5 × 10(-11)) and KLK2 (R(2) = 0.5893, P < 5 × 10(-10)) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression.